Wigenstam, Elisabeth

Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine.

2012 (English)Doctoral thesis, comprehensive summary (Other academic)

Abstract [en]

Inhalation of chemical substances can cause irritation to airways and in high doses acute airway injury. When mice are exposed to the alkylating nitrogen mustard analogue melphalan they develop an acute airway inflammation with a rapid influx of neutrophils to the lungs. The acute phase is followed by long-term respiratory complications characterized by bronchitis, lung fibrosis, and airway hyperreactivity.

In this thesis, a mouse model for chemical airway inflammation was established and the effects on the lungs in a time span from 6 hours up to 3 months were investigated in order to study both acute effects and possible chronic injury. We find that treatment with corticosteroids, e.g. dexamethasone, effectively blocks the inflammatory reaction in several ways: Neutrophil influx to the lungs is diminished, the expression of the proinflammatory cytokines interleukin (IL) -6 and IL-1b is decreased and edema formation as well as development of lung fibrosis is mitigated. In acute airway inflammation we show that the antioxidant vitamin E can be used as a possible complement to corticosteroids but not as a replacement since it causes insufficient downregulation of the inflammatory response. We show the importance of the T lymphocytes as they play a prominent role in the pathogenesis of long-term lung injuries caused by melphalan. Especially the minor gd T cell subset is of major importance orchestrating a number of responses including the acute cytokine and neutrophil response and late-phase lung fibrosis.

In order to find the critical time for dexamethasone treatment, mice were exposed to melphalan, treated with dexamethasone at specific time points and lung physiology and airway reactivity was measured in anaesthetized, tracheostomized mice using a small animal ventilator. From these results we conclude that an early treatment, i.e. within one hour after exposure, with dexamethasone is needed to prevent chronic lung injury.

This thesis was undertaken with the main goal to better understand the pathogenesis of melphalan-induced airway inflammation. We believe that our findings have shed new light in this area of research and hope that this increased knowledge may be of future clinical use.

Abstract [en]

The pathogenesis of lung injury by exposure to highly toxic sulfur and nitrogen mustards involves alkylating damage of the respiratory epithelium followed by an acute inflammatory response and lung edema. The acute phase is followed by long-term respiratory complications characterized by bronchitis, lung fibrosis, and airway hyperreactivity. In this study, we utilized a mouse model for airway inflammation induced by inhalation exposure to the alkylating nitrogen mustard melphalan, in order to investigate possible beneficial treatment effects by the corticosteroid dexamethasone. In addition, we investigated therapeutic efficacy of liposome-encapsuled vitamin E, an antioxidant formulation previously shown to be efficient in counteracting inflammatory conditions. Influx of inflammatory cells to airways, edema formation, and expression of different cytokines were analyzed 6 and 18 hours after exposure to melphalan. In order to evaluate long-term lung effects, we also investigated collagen deposition and accumulation of lymphocytes at 2 and 4 weeks after exposure. A single intraperitoneal injection of dexamethasone (10 mg/kg body weight) 1 hour after melphalan exposure significantly reduced interleukin (IL)-1 and IL-6 in bronchoalveolar lavage fluid (BALF) and diminished the acute airway inflammation. Our results also indicate that early single-dose treatment with dexamethasone protects against long-term effects observed 2-4 weeks after melphalan exposure, as indicated by reduced lymphocytic response in airways and decreased collagen deposition. Furthermore, our results indicate that also vitamin E (50 mg/kg) reduces acute inflammatory cell influx, and suppresses collagen formation in lung tissue, indicating that this drug could be used in combination with corticosteroids for protection against chemical-induced lung injury.

Abstract [en]

Low-dose exposure of alkylating mustard gas causes long-term respiratory complications characterized by bronchitis and lung fibrosis. In this study, we utilized a mouse model for lung exposure of the nitrogen mustard melphalan, in order to define early and late events in the pathogenesis such as expression of pro-inflammatory cytokines, recruitment of inflammatory cells to airways and late-phase fibrosis. We investigated the roles of different T lymphocyte subsets on the inflammatory response by using knockout mice lacking either the genes expressing T cell receptor (TCR)αβ or TCRγδ, and compared the responsiveness with that of wild type mice and double knockout mice completely deficient in T cells. Exposure to melphalan induced an early burst of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6 and IL-23 in airways, followed by extensive infiltration of neutrophils in the lung tissue and airways within 24h. The acute phase was followed by a sustained lymphocytic response that persisted for at least 14 days with resulting lung fibrosis. Engagement of T lymphocytes, particularly the γδ T cell subset, was crucial both for the acute cytokine and neutrophil response and for the late-phase lung fibrosis as indicated by the lack of response in γδ T cell deficient mice. Our data demonstrate that T lymphocytes play a prominent role in the pathogenesis of long-term lung injuries caused by strong alkylating agents.

Bucht, Anders

Abstract [en]

Context: Exposure to toxic alkylating mustard agents causes both acute and long-term effects to the lungs as indicated by increased number of inflammatory cells in airways, lung edema and lung tissue fibrosis. We have previously demonstrated that treatment with the corticosteroid dexamethasone 1 hr after lung exposure to the alkylating mustard melphalan, protect mice from acute and sub-acute inflammatory responses, as well as from lung fibrosis.

Objective: In order to address the importance of early anti-inflammatory treatment, we investigated the therapeutic effect of dexamethasone administered 1, 2 or 6 hrs following exposure to melphalan.

Methods: Female C57BL/6 mice were via intratracheal instillation exposed to the nitrogen mustard analogue melphalan and treated i.p. with dexamethasone 1, 2 or 6 hours after exposure. Twenty hours or 14 days post exposure mice were subjected to analysis of respiratory mechanics where the effects of incremental doses of methacholine on central and peripheral lung components were measured. We also determined the amount of neutrophils and lymphocytes in the bronchoalveolar lavage fluid and measured the amount of collagen content in the lungs.

Results: Melphalan exposure exerted a significant effect on both central and peripheral respiratory function. Dexamethasone given one hour post exposure protected the lung against the damaging effects of melphalan. Collagen deposition 14 days after exposure was decreased with dexamethasone treatment.

Conclusion: Early dexamethasone treatment (within one hour after exposure) is important in order to reduce the airway reactivity and inflammation caused by toxic alkylating mustards such as melphalan.

Pejler, Gunnar

Bucht, Anders

Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine.

(English)Manuscript (preprint) (Other academic)

Abstract [en]

Background: Mammals are incapable of producing chitin but express despite this, enzymes such as chitinases and chitinase-like proteins that are involved in the regulation of its biosynthesis. There is increasing evidence, in both human and mice, that chitinases and chitinase-like proteins are important mediators of immune responses. Studies show that two chitinase-like proteins, Ym1 and Ym2, are expressed in murine models of allergen-induced lung inflammation. The purpose of this study was to investigate whether Ym1 and Ym2 are specific markers for allergic inflammation or if they were to some extent expressed in other inflammatory settings as well.

Methods: In this study, three different models for airway inflammation using C57BL/6 female mice were utilized. We induced allergic airway inflammation with a 35-day protocol using ovalbumin; chemical airway inflammation by intratracheal exposure of the alkylating nitrogen mustard analogue melphalan; and endotoxin-induced pulmonary inflammation by exposure to aerosolized lipopolysaccharide. Twenty hours after final exposure/challenge, lung tissue and cells in bronchoalveolar lavage were analyzed. Transcription of Ym1 and Ym2 mRNA was determined using real-time reverse-transcription PCR and protein expression was analyzed with 2D gel electrophoresis.

Results and conclusion: We demonstrated that both Ym1 and Ym2 are specifically up-regulated in an allergic airway inflammation but not in LPS-induced or melphalan-induced airway inflammation. Based on our results we consider Ym2 a possible future candidate as a specific marker for allergic airway inflammation.